Methods and compositions for inactivating alpha 1,6 fucosyltransferase (fut8) gene expression

ABSTRACT

Disclosed herein are methods and compositions for inactivating a FUT8 gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 12/931,265, filed Jan. 27, 2011, now allowed, whichis a divisional application of U.S. patent application Ser. No.12/218,035, filed Jul. 10, 2008, now U.S. Pat. No. 7,919,313, whichclaims the benefit of U.S. Provisional Application No. 60/959,202, filedJul. 12, 2007 and U.S. Provisional Application No. 60/993,624, filedSep. 13, 2007, the disclosures of which are hereby incorporated byreference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of genome engineering, cellculture and protein production.

BACKGROUND

Monoclonal antibody therapy is a large and growing treatment modality inmedicine (Glennie et al. (2000) Immunol Today 21:403-10). There are morethan twenty FDA-approved monoclonal antibody therapies, with many morecurrently in clinical trials. Antibody therapy directed against solublefactors (such as vascular endothelial growth factor or tumor necrosisfactor, e.g.), aims simply to reduce the free ligand concentration byimmunocomplex formation. In contrast, when antibody therapy is directedat cell surface antigens (as is often the case in anti-neoplasticimmunotherapy), the goal is often the removal of the cell itself. Thetherapeutic antibody may induce apoptosis directly (Shan et al. (1998)Blood 91:1644-52; Shan (2000) Cancer Immunol Immunother 48:673-83), butmore often it must recruit the patient's immune system to destroy thetarget cell. See, Reff et al. (1994) Blood 83:435-45; Idusogie et al.(2000) J Immunol 164:4178-84; Golay et al. (2000) Blood 95:3900-8;Harjunpaa et al. (2000) Scand J Immunol 51:634-41; Anderson et al.(1997) Biochem Soc Trans 25, 705-8; Clynes et al. (1998) Proc Natl AcadSci USA 95:652-6; Clynes et al. (2000) Nat Med 6: 443-6; Sampson et al.(2000) Proc Natl Acad Sci USA 97:7503-8.

There are two main mechanisms by which the antibody-activated immunesystem can destroy offending cells: complement-dependent cytotoxicity(CDC) and antibody-dependent cellular cytotoxicity (ADCC). ADCC is animmune response generated primarily by natural killer (NK) cells againstantibody-coated targets. See, Lewis et al. (1993) Cancer ImmunolImmunother 37:255-63. In ADCC, NK cells recognize the constant (Fc)region of antibodies primarily via interaction with the NK cell'sFcγRIII receptor. The NK cells then deposit perforins and granzymes onthe target cell surface inducing cell lysis and apoptosis, respectively.The Fc-FcγRIII interaction is extremely sensitive to Fc glycosylation.Aglycosylated immunoglobulins fail to bind Fc receptors. See,Leatherbarrow et al. (1985) Mol Immunol 22:407-15 (1985); Walker et al.(1989) Biochem J259:347-53 (1989); Leader et al. (1991) Immunology72:481-5. In addition, fucosylation of the carbohydrate chain attachedto Asn297 of the Fc region inhibits binding to FcγRIII and reduces invitro ADCC activity. See, Shields et al. (2002) J Biol Chem277:26733-40; Shinkawa et al. (2003) J Biol Chem 278:3466-73; Niwa etal. (2004) Cancer Res 64:2127-33.

The majority of mammalian immunoglobulins are fucosylated, includingthose produced in Chinese hamster ovary cells (CHO cells, Cricetulusgriseus). Jefferis et al. (1990) Biochem J 268:529-37; Hamako et al.(1993) Comp Biochem Physiol B 106:949-54; Raju et al. (2000)Glycobiology 10:477-86. Fucose attachment to the Fc core region is viaan α-1,6 linkage generated by the α1,6 fucosyltransferase (Fut8)protein, apparently the sole α-1,6 fucosyltransferase in mammaliancells. Oriol et al. (1999) Glycobiology 9:323-34; Costache et al. (1997)J Biol Chem 272:29721-8. Disruption of the FUT8 gene in CHO cellseliminated core fucosylation of antibodies and increased ADCC by around100-fold. See, Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22).However, such conventional gene disruption by homologous recombinationis typically a laborious process. This was particularly true in the caseof C. griseus FUT8, as approximately 120,000 clonal cell lines werescreened to discover three healthy FUT8 −/− clones (Yamane-Ohnuki et al.(2004), supra).

Thus, there remains a need for cells lines in which Fut8 expression ispartially or fully inactivated. Site-specific cleavage of genomic locioffers an efficient supplement and/or alternative to conventionalhomologous recombination. Creation of a double-strand break (DSB)increases the frequency of homologous recombination at the targetedlocus more than 1000-fold. More simply, the inaccurate repair of asite-specific DSB by non-homologous end joining (NHEJ) can also resultin gene disruption. Creation of two such DSBs can result in deletion ofarbitrarily large regions. The modular DNA recognition preferences ofzinc fingers protein allows for the rational design of site-specificmulti-finger DNA binding proteins. Fusion of the nuclease domain fromthe Type II restriction enzyme Fok I to site-specific zinc fingerproteins allows for the creation of site-specific nucleases. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275, the disclosures of which are incorporated byreference in their entireties for all purposes.

SUMMARY

Disclosed herein are compositions for the partial or completeinactivation of a FUT8 gene. Also disclosed herein are methods of makingand using these compositions (reagents), for example to inactivate FUT8in a cell to produce cell lines in which a FUT8 gene is inactivated.FUT8 disrupted cell lines are useful, for example, in production ofrecombinant proteins such as α1-antitrypsin and monoclonal antibodies asantibodies produced in cells where Fut8 expression is reduced exhibitenhanced ADCC function.

In one aspect, zinc finger proteins, engineered to bind in a FUT8 gene,are provided. Any of the zinc finger proteins described herein mayinclude 1, 2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in a FUT8 gene. Incertain embodiments, the zinc finger proteins comprise 4, 5 or 6 fingers(wherein the individual zinc fingers are designated F1, F2, F3, F4, F5and F6) and comprise the amino acid sequence of the recognition helicesshown in Table 1.

In certain embodiments, provided herein is an engineered zinc fingerprotein DNA-binding domain, wherein the DNA-binding domain comprisesfour zinc finger recognition regions ordered F1 to F4 from N-terminus toC-terminus, and wherein F1, F2, F3, and F4 comprise the following aminoacid sequences: F1: QSSDLSR (SEQ ID NO:9); F2: TSGNLTR (SEQ ID NO:10);F3: RSDDLSK (SEQ ID NO:11); and F4: DRSALAR (SEQ ID NO:12).

In other embodiments, the disclosure provides an engineered zinc fingerprotein DNA-binding domain, wherein the DNA-binding domain comprisesfour zinc finger recognition regions ordered F1 to F4 from N-terminus toC-terminus, and wherein F1, F2, F3, and F4 comprise the following aminoacid sequences: F1: RSDVLSA (SEQ ID NO:14); F2: QNATRIN (SEQ ID NO:15);F3: DRSNLSR (SEQ ID NO:16); and F4: RLDNRTA (SEQ ID NO:17).

In other embodiments, the disclosure provides an engineered zinc fingerprotein DNA-binding domain, wherein the DNA-binding domain comprises sixzinc finger recognition regions ordered F1 to F6 from N-terminus toC-terminus, and wherein F1, F2, F4, F5 and F6 comprise the followingamino acid sequences: F1: RSDNLSV (SEQ ID NO:19); F2: QNATRIN (SEQ IDNO:15); F4: QSATRTK (SEQ ID NO:21); F5 RSDNLSR (SEQ ID NO:22); and F6:RNDNRKT (SEQ ID NO:23). In any of these embodiments, F3 may compriseRSDNLST (SEQ ID NO:20) or RSDHLSQ (SEQ ID NO:24).

In other embodiments, the disclosure provides an engineered zinc fingerprotein DNA-binding domain, wherein the DNA-binding domain comprisesfive zinc finger recognition regions ordered F1 to F5 from N-terminus toC-terminus, and wherein F1, F2, F3, F4, and F5 comprise the followingamino acid sequences: F1: RSDNLRE (SEQ ID NO:26); F2: NNTQLIE (SEQ IDNO:27); F3: TSSILSR (SEQ ID NO:28); F4 RSDNLSA (SEQ ID NO:29); and F5:RKDTRIT (SEQ ID NO:30).

In another aspect, fusion proteins comprising any of the zinc fingerproteins described herein and at least one cleavage domain or at leastone cleavage half-domain, are also provided. In certain embodiments, thecleavage half-domain is a wild-type FokI cleavage half-domain. In otherembodiments, the cleavage half-domain is an engineered FokI cleavagehalf-domain.

In yet another aspect, a polynucleotide encoding any of the proteinsdescribed herein is provided.

In yet another aspect, also provided is an isolated cell comprising anyof the proteins and/or polynucleotides as described herein. In certainembodiments, Fut8 is inactivated (partially or fully) in the cell. Anyof the cells described herein may include additional genes that havebeen inactivated, for example, using zinc finger nucleases designed tobind to a target site in the selected gene. In certain embodiments,provided herein are cells or cell lines in which FUT8, dihydrofolatereductase (DHFR) and glutamine synthetase (GS) have been inactivated.

In addition, methods of using the zinc finger proteins and fusionsthereof in methods of inactivating FUT8 in a cell or cell line areprovided. In certain embodiments, inactivating a FUT8 gene results in acell line which can produce recombinant proteins at higher levels or inwhich one or more activities (functions) of the proteins are increasedas compared to proteins produced in cells where the FUT8 gene is notinactivated. For example, cell lines as described herein having aninactivated FUT8 gene can be used to produce monoclonal antibodies thatexhibit enhanced ADCC function for immunotherapy. Cell lines asdescribed herein can also be used to produce recombinant α1-antitrypsin.

Thus, in another aspect, provided herein is a method for inactivating acellular FUT8 gene (e.g., an endogenous FUT8 gene) in a cell, the methodcomprising: (a) introducing, into a cell, a first nucleic acid encodinga first polypeptide, wherein the first polypeptide comprises: (i) a zincfinger DNA-binding domain that is engineered to bind to a first targetsite in an endogenous FUT8 gene; and (ii) a cleavage domain; such thatthe polypeptide is expressed in the cell, whereby the polypeptide bindsto the target site and cleaves the FUT8 gene. In certain embodiments,the methods further comprise introducing a nucleic acid encoding asecond polypeptide, wherein the second polypeptide comprises: (i) a zincfinger DNA-binding domain that is engineered to bind to a second targetsite in the FUT8 gene; and (ii) a cleavage domain; such that the secondpolypeptide is expressed in the cell, whereby the first and secondpolypeptides bind to their respective target sites and cleave the FUT8gene. The first and second polypeptides may be encoded by the firstnucleic acid or by different nucleic acids. In certain embodiments, oneor more additional polynucleotides or polypeptides are introduced intothe cells, for example polynucleotides encoding additional zinc fingerproteins.

In yet another aspect, the disclosure provides a method of producing arecombinant protein of interest in a host cell, the method comprisingthe steps of: (a) providing a host cell comprising an endogenous FUT8gene; (b) inactivating the endogenous FUT8 gene of the host cell by anyof the methods described herein; and (c) introducing an expressionvector comprising a transgene, the transgene comprising a sequenceencoding a protein of interest, into the host cell, thereby producingthe recombinant protein. In certain embodiments, the protein of interestcomprises an antibody, e.g., a monoclonal antibody.

In any of the cells and methods described herein, the cell or cell linecan be a COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX,CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0, SP2/0-Ag14,HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6, insect cellsuch as Spodoptera fugiperda (Sf), or fungal cell such as Saccharomyces,Pichia and Schizosaccharomyces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of C. griseus FUT8 cDNA sequence(SEQ ID NO:1).

FIG. 2 depicts the amino acid sequence of C. griseus Fut8 (SEQ ID NO:2).

FIG. 3 depicts partial nucleotide sequence of exon 9, intron 9, exon 10and intron 10 of C. griseus FUT8 genomic DNA (SEQ ID NO:3).

FIG. 4, panels A and B, are schematics depicting the location of zincfinger nuclease (ZFN) binding and cleavage sites within FUT8 exon 10.FIG. 4A is an overview of the exon 9-intron 10 region. Exons aredepicted with black arrows and the grey line shows non-coding DNA. FIG.4B shows a detailed view of the fucosyltransferase motif II and ZFNbinding sites. The location of the fucosyltransferase motif II (lightgrey box) was determined as described in Oriol et al. (1999)Glycobiology 9:323-34 (1999). The translation of the Fut motif II isshown above the DNA sequence (SEQ ID NOS: 65 and 66, respectively). Thelocation of the recognition sequences of the ZFNs in relation to thesense strand of the gene are shown as dark grey boxes. ZFN 12176(Table 1) is a five Zn-finger protein recognizing a 15 bp target siteand ZFN 12170 (Table 1) is a six Zn-finger protein recognizing 18 bptarget site. The last two nucleotides shown (GT) are the 5′ splice donorsite for intron 10.

FIG. 5, panels A and B, show results of Cel-1 mismatch assays for ZFNactivity at the endogenous FUT8 locus. The efficacy of each ZFN pair isreflected in the total amount of cleavage products beneath the parentPCR product. FIG. 5A shows Cel-1 assays results for DNA harvested twodays post-transfection of the indicated plasmids and a portion of theFUT8 locus PCR amplified using the oligos GJC 71F: GCTTGGCTTCAAACATCCAG(SEQ ID NO:4) and GJC 89R: GGACTTGTTCCGTGTGTTCT (SEQ ID NO:5). The sizesof the predicted cleavage products are 264 bp and 216 bp. FIG. 5B showsresults of DNA was harvested two days post-transfection and a portion ofthe FUT8 locus PCR amplified using the oligos GJC 90F:CTGTTGATTCCAGGTTCCCA (SEQ ID NO:6) and GJC 91R: TGTTACTTAAGCCCCAGGC (SEQID NO:7). The sizes of the predicted cleavage products are 431 bp and342 bp. ZFN combinations are shown above the appropriate lanes;ZFN-specific cleavage products are indicated with white arrowheads. Thepercent of chromosomes modified by non-homologous end joining is listedbelow each lane. The size of molecular weight marker bands is indicatedto the left of the gel.

FIG. 6, panels A and B, depict ZFN activity at the endogenous FUT8locus. FIG. 6A shows results of nuclease-mediated deletion of 1.3 kb ofFUT8 using the two indicated ZFN pairs, which were transfected inparallel, with the genomic DNA harvested two days later. Deletion of the˜1300 bp between the ZFN sites generates a ˜559 bp product. FIG. 6Bresults of Cel-1 mismatch assays for ZFN activity at the endogenous FUT8locus with and without Lens culinaris agglutinin (LCA) enrichment in CHOcells. ZFN pairs are shown at the top line (ZFN pair 12170 and 12176 forlanes 2 to 4 (from left to right) and ZFN pair 12172 and 12176 for lanes5 to 7 (from left to right)). The lane designated “pVAX” refers tocontrol cells transfected with empty plasmid (no ZFN). The lanedesignations “2” and “30” refer to days post-transfection and “LCA”refers to cells subject to LCA selection.

FIG. 7 shows results of Cel-1 mismatch assays for ZFN activity at theendogenous FUT8 locus with and without Lens culinaris agglutinin (LCA)enrichment in CHO cells in which both the dihydrofolate reductase (DHFR)and glutamine synthetase (GS) genes have been disrupted by previous ZFNtreatment. ZFN pools (1, 3, 5, 7) used are shown at the top of each laneand the percent of chromosomes modified by non-homologous end joining islisted below each lane. The two lanes marked “Cont.” show negativecontrols in which cells were transfected with a GFP expression plasmid(left “Cont.” lane) or untransfected (right “Cont.” lane).

FIG. 8 shows the indicated characteristics of triple gene (DHFR/GS/FUT8)knockout clones isolated from LCA-enriched ZFN-treated pools #1.

FIG. 9 is a graph depicting binding of fluorescent LCA to the indicatedcell types. “Unstained” refers to CHO cells that contain wild type Fut8but which have not been exposed to fluorescent LCA; “triple KO” refersto cells in which all three of the DHFR, GS and FUT8 genes have beendisrupted using ZFNs; and “fut8 wt” refers to CHO cells that containwild type FUT8 and have been exposed to fluorescent LCA.

FIG. 10 shows results of Cel-1 mismatch assays for ZFN activity at theendogenous FUT8 locus in FUT8 hypomorphs. Clone number is indicatedabove the lane and allele disruptions are indicated below each lane.

FIG. 11 is a graph depicting binding of fluorescent LCA to the indicatedcell types. “Wild type no stain” refers to CHO cells that contain wildtype Fut8 but which have not been exposed to fluorescent LCA;“Hypomorphic clone” refers to a population of cells in which FUT8 waspartially inactivated (FUT8 hypomorph); and “CHO-K1” refers to CHO cellsthat contain wild type FUT8 and have been exposed to fluorescent LCA.

DETAILED DESCRIPTION

Described herein are compositions and methods for partial or completeinactivation of a FUT8 gene. Also disclosed are methods of making andusing these compositions (reagents), for example to inactivate a Fut8gene in a target cell. Inactivation of Fut8 in a target cell can be usedto produce cell lines for expression of recombinant proteins,particularly monoclonal antibodies that elicit enhanced ADCC.

In mammalian cells, Fut8 attaches core fucose to the oligosaccharidespresent on the Fc region of antibodies, which is widely recognized asbeing of great importance for the effector function ofantibody-dependent cellular cytotoxicity. Three-dimensional analysis ofthe structure of human Fut8 has shown that the three α2/α6fucosyltransferase motifs form the catalytic core of the enzyme. See,Ihara et al. (2007) Glycobiology 17:455-66. In this region, pointmutation of many single residues to alanine results in completeinactivation of the enzyme. See, Ihara et al. (2007) Glycobiology17:455-66; Takahashi et al. (2000) Glycobiology 10:503-10. As notedabove, cells in which FUT8 expression is reduced or eliminated (e.g.knockout cell lines or with siRNA), produce non-fucosylated antibodiesthat have greater effector function. See, e.g., Kanada et al. (2007)Biotechnol. 130(3):300-310; Kanada et al. (2007) Glycobiology18:104-118; Mori et al. (2004) Biotechnol. Bioeng. 88:901-908.

Thus, the methods and compositions described herein provide a highlyefficient method for targeted gene knockout (partial or complete) ofFUT8 that allows for the generation of cells lines for producingnon-fucosylated proteins such as antibodies.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolfe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing infoiniation in a databasestoring information of existing ZFP designs and binding data. See, forexample, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. No.5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat.No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found on theGenBank website. With respect to sequences described herein, the rangeof desired degrees of sequence identity is approximately 80% to 100% andany integer value therebetween. Typically the percent identities betweensequences are at least 70-75%, preferably 80-82%, more preferably85-90%, even more preferably 92%, still more preferably 95%, and mostpreferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that fauns between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

An “cleavage half-domain” is a polypeptide sequence which, inconjunction with a second polypeptide (either identical or different)forms a complex having cleavage activity (preferably double-strandcleavage activity). The terms “first and second cleavage half-domains;”“+ and − cleavage half-domains” and “right and left cleavagehalf-domains” are used interchangeably to refer to pairs of cleavagehalf-domains that dimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain) See, also,U.S. Patent Publication Nos. 2005/0064474; 2007/0218528 and2008/0131962, incorporated herein by reference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases. An exogeneous molecule can also be the same type of moleculeas an endogenous molecule but derived from a different species than thecell is derived from. For example, a human nucleic acid sequenced may beintroduced into a cell line originally derived from a mouse or hamster.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, shRNAs, micro RNAs (miRNAs) ribozyme, structural RNA or any othertype of RNA) or a protein produced by translation of a mRNA. Geneproducts also include RNAs which are modified, by processes such ascapping, polyadenylation, methylation, and editing, and proteinsmodified by, for example, methylation, acetylation, phosphorylation,ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Gene inactivation refers to anyreduction in gene expression as compared to a cell that does not includea ZFP as described herein. Thus, gene inactivation may be complete(knock-out) or partial (e.g., a hypomorph in which a gene exhibits lessthan normal expression levels or a product of a mutant gene that showspartial reduction in the activity it influences).

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:hybrid (chimeric) antibody molecules (see, for example, Winter et al.,Nature (1991) 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 andF(ab) fragments; Fv molecules (non-covalent heterodimers, see, forexample, Inbar et al., Proc Natl Acad Sci USA (1972) 69:2659-2662; andEhrlich et al., Biochem (1980) 19:4091-4096); single-chain Fv molecules(sFv) (see, for example, Huston et al., Proc Natl Acad Sci USA (1988)85:5879-5883); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al., Biochem (1992) 31:1579-1584; Cumberet al., J Immunology (1992) 149B: 120-126); humanized antibody molecules(see, for example, Riechmann et al., Nature (1988) 332:323-327;Verhoeyan et al., Science (1988) 239:1534-1536; and U.K. PatentPublication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the teen “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and other fragments, as well as chimeric and humanizedhomogeneous antibody populations that exhibit immunological bindingproperties of the parent monoclonal antibody molecule.

Zinc Finger Nucleases

Described herein are zinc finger nucleases (ZFNs) that can be used forinactivation of a FUT8 gene. ZFNs comprise a zinc finger protein (ZFP)and a nuclease (cleavage) domain.

A. Zinc Finger Proteins

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentApplication Publication Nos. 20050064474 and 20060188987, incorporatedby reference in their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length (e.g., TGEKP (SEQ ID NO:36),TGGQRP (SEQ ID NO:37), TGQKP (SEQ ID NO:38), and/or TGSQKP (SEQ IDNO:39)). See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949for exemplary linker sequences 6 or more amino acids in length. Theproteins described herein may include any combination of suitablelinkers between the individual zinc fingers of the protein. Examples ofadditional linker structures are found in U.S. Provisional ApplicationNo. 61/130,099, filed May 28, 2008 and entitled Compositions For LinkingDNA-Binding Domains And Cleavage Domains.

Table 1 describes a number of zinc finger binding domains that have beenengineered to bind to nucleotide sequences in the FUT8 gene. See, also,FIG. 1 and FIG. 3. Each row describes a separate zinc finger DNA-bindingdomain. The DNA target sequence for each domain is shown in the firstcolumn and the second through fifth columns show the amino acid sequenceof the recognition region (amino acids −1 through +6, with respect tothe start of the helix) of each of the zinc fingers (F1 through F4, F5or F6) in the protein. Also provided in the first column is anidentification number for the proteins.

TABLE 1 Zinc finger nucleases targeted to Fut8 ZFN name Target sequenceF1 F2 F3 F4 F5 F6 ZFN 12029 QSSDLSR TSGNLTR RSDDLSK DRSALAR N/A N/AGTCTCGGATGCT (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 9)NO: 10) NO: 11) NO: 12) ZFN 12030 RSDVLSA QNATRIN DRSNLSR RLDNRTA N/AN/A AAGGACACACTG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 13) NO: 14)NO: 15) NO: 16) NO: 17) ZFN 12170 RSDNLSV QNATRIN RSDNLST QSATRTKRSDNLSR RNDNRKT AAGGAGGCAAAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID ACAAAG NO: 19) NO: 15) NO: 20) NO: 21) NO: 22) NO: 23)(SEQ ID NO: 18) ZFN 12172 RSDNLSV QNATRIN RSDHLSQ QSATRTK RSDNLSRRNDNRKT AAGGAGGCAAAG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDACAAAG NO: 19) NO: 15) NO: 24) NO: 21) NO: 22) NO: 23) (SEQ ID NO: 18)ZFN 12176 RSDNLRE NNTQLIE TSSILSR RSDNLSA RKDTRIT AAGAAGGGTCATCAG(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID N/A (SEQ ID NO: 25) NO: 26)NO: 27) NO: 28) NO: 29) NO: 30)

As described below, in certain embodiments, a four-, five-, orsix-finger binding domain as shown in Table 1 is fused to a cleavagehalf-domain, such as, for example, the cleavage domain of a Type IIsrestriction endonuclease such as FokI. One or more pairs of such zincfinger/nuclease half-domain fusions are used for targeted cleavage, asdisclosed, for example, in U.S. Patent Publication Nos. 20050064474 and20070218528.

For targeted cleavage, the near edges of the binding sites can separatedby 5 or more nucleotide pairs, and each of the fusion proteins can bindto an opposite strand of the DNA target. All pairwise combinations(e.g., ZFN 12029 with ZFN 12030, and ZFN 12170 with either ZFN 12172 orZFN 12176) of the proteins shown in Table 1 can be used for targetedcleavage of a FUT8 gene. Following the present disclosure, ZFNs can betargeted to any sequence in a FUT8 gene.

B. Cleavage Domains

The ZFNs also comprise a nuclease (cleavage domain, cleavagehalf-domain). The cleavage domain portion of the fusion proteinsdisclosed herein can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which a cleavage domain can be derivedinclude, but are not limited to, restriction endonucleases and homingendonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme Fok I catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. See, for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and5,487,994; as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768;Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al.(1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment,fusion proteins comprise the cleavage domain (or cleavage half-domain)from at least one Type IIS restriction enzyme and one or more zincfinger binding domains, which may or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95: 10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the Fok I enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fok I fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and two FokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fok Ifusions are provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished when one or morepairs of nucleases containing these cleavage half-domains are used forcleavage. See, e.g., U.S. Patent Publication No. 20080131962, thedisclosure of which is incorporated by reference in its entirety for allpurposes.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474 (Example 5) and 20070134796 (Example 38).

C. Additional Methods for Targeted Cleavage in FUT8

Any nuclease having a target site in a FUT8 gene can be used in themethods disclosed herein. For example, homing endonucleases andmeganucleases have very long recognition sequences, some of which arelikely to be present, on a statistical basis, once in a human-sizedgenome. Any such nuclease having a unique target site in a FUT8 gene canbe used instead of, or in addition to, a zinc finger nuclease, fortargeted cleavage in a FUT8 gene.

Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce,I-SceIV, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Theirrecognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S.Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res.25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994)Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228;Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast et al. (1998) J.Mol. Biol. 280:345-353 and the New England Biolabs catalogue.

Although the cleavage specificity of most homing endonucleases is notabsolute with respect to their recognition sites, the sites are ofsufficient length that a single cleavage event per mammalian-sizedgenome can be obtained by expressing a homing endonuclease in a cellcontaining a single copy of its recognition site. It has also beenreported that the specificity of homing endonucleases and meganucleasescan be engineered to bind non-natural target sites. See, for example,Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003)Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66.

Delivery

The ZFNs described herein may be delivered to a target cell by anysuitable means. Suitable cells include but not limited to eukaryotic andprokaryotic cells and/or cell lines. Non-limiting examples of such cellsor cell lines include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,HaK, NS0, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),and perC6 cells as well as insect cells such as Spodoptera fugiperda(Sf), or fungal cells such as Saccharomyces, Pichia andSchizosaccharomyces.

Methods of delivering proteins comprising zinc fingers are described,for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261;6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824, the disclosures of all of which areincorporated by reference herein in their entireties.

FUT8 ZFNs as described herein may also be delivered using vectorscontaining sequences encoding one or more of the ZFNs. Any vectorsystems may be used including, but not limited to, plasmid vectors,retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirusvectors; herpesvirus vectors and adeno-associated virus vectors, etc.See, also, U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113;6,979,539; 7,013,219; and 7,163,824, incorporated by reference herein intheir entireties. Furthermore, it will be apparent that any of thesevectors may comprise one or more ZFN encoding sequences. Thus, when oneor more pairs of ZFNs are introduced into the cell, the ZFNs may becarried on the same vector or on different vectors. When multiplevectors are used, each vector may comprise a sequence encoding one ormultiple ZFNs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology Doerfler and Böhm (eds.) (1995); and Yuet al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding engineered ZFPsinclude electroporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.)and Copernicus Therapeutics Inc., (see for example U.S. Pat. No.6,008,336).

Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No.4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents aresold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Feigner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Vivol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression of a ZFP fusion protein ispreferred, adenoviral based systems can be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand high levels of expression have been obtained. This vector can beproduced in large quantities in a relatively simple system.Adeno-associated virus (“AAV”) vectors are also used to transduce cellswith target nucleic acids, e.g., in the in vitro production of nucleicacids and peptides, and for in vivo and ex vivo gene therapy procedures(see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No.4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994);Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinantAAV vectors are described in a number of publications, including U.S.Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260(1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat& Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Tad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NS0,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pichia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used.

Applications

The disclosed methods and compositions can be used for inactivation of aFUT8 genomic sequence. As noted above, inactivation includes partial orcomplete repression of FUT8 gene expression in a cell. Inactivation of aFUT8 gene can be achieved, for example, by a single cleavage event, bycleavage followed by non-homologous end joining, by cleavage at twosites followed by joining so as to delete the sequence between the twocleavage sites, by targeted recombination of a missense or nonsensecodon into the coding region, by targeted recombination of an irrelevantsequence (i.e., a “stuffer” sequence) into the gene or its regulatoryregion, so as to disrupt the gene or regulatory region, or by targetingrecombination of a splice acceptor sequence into an intron to causemis-splicing of the transcript.

There are a variety of applications for ZFN-mediated inactivation(knockout or knockdown) of FUT8. For example, the methods andcompositions described herein allow for the generation of Fut8-deficientcell lines for use in recombinant protein production, for exampleα1-antitrypsin and/or monoclonal antibody production. Cells in whichFut8 is inactivated produce antibodies that exhibit greater effectorfunction, particularly in the induction of ADCC.

Similarly, cells in which Fut8 is partially or completely inactivatedcan also be used to produce the fucosylated serine protease inhibitoralpha 1-Antitrypsin or α1-antitrypsin (A1AT). A1AT may play a role inthe inhibition of cancer metastasis (Goodarzi and Turner (1995) ChimActo 236(2): 161-171) and patients afflicted with a variety of cancersexhibit A1AT which is more heavily fucosylated as compared to that foundin the normal population (Saitoh et al. (1993) Archives Biochem. &Biophysics 303:281-287), suggesting that fucosylation of the endogenousA1AT may lead to decreased functionality. In addition, it has been shownthat the presence of fucosylated A1AT in ovarian cancer patients ispredictive of unresponsiveness to chemotherapy (Thompson et al. (1988)Br. J. Cancer 58(5):89-93). Alpha 1-antitrypsin isolated from bloodplasma has been used for the treatment of lung degradation (e.g.,pulmonary emphysema) caused by a lack of A1AT. Production of A1AT in afut8 knockout or knockdown cell line could yield protein with greaterconsistency and activity. Thus, the cells and cell lines describedherein also allow for the efficient production of A1AT.

Additional genes in the Fut8 deficient cells and cells lines describedherein may also be inactivated. Additional genes involved in proteinoverexpression as well as compositions and methods for inactivatingthese genes are disclosed in U.S. Patent Publication Nos. 2006/0063231and 20080015164, the disclosures of which are incorporated by referencein their entireties herein. For instance, as disclosed in Example 5,cells can be generated using the methods described herein in which Fut8,dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have beeninactivated. See, Example 5. These cells are useful for overexpressing aprotein of interest.

EXAMPLES Example 1 Design and Construction of FUT8 ZFNs

Three motifs conserved across α-2 and α-6 fucosyltransferases areresponsible for enzymatic activity of Fut8 and subsequent fucosylationof recombinantly produced antibody therapeutics. See, Ihara et al.(2007) Glycobiology 17:455-66. These motifs were readily identifiable inthe hamster (C. griseus) sequence. See, Oriol et al. (1999) Glycobiology9:323-34; Javaud et al. (2000) Mol Biol Evol 17:1661-72. In particular,the hamster FUT8 Fut motif II (FIG. 4) is identical to the cow and humanmotifs, and only one amino acid different than those from pig and mouse.Javaud et al. (2000) Mol Biol Evol 17:1661-72. In addition, alignment ofM. musculus and R. norvegicus FUT8 genomic DNA sequence suggested thatintron 9 of the C. griseus FUT8 gene would be small enough to be readilyclonable.

The C. griseus FUT8 cDNA was cloned as follows. Ten nanograms of a cDNAlibrary derived from CHO-S cells was PCR amplified using primers GJC119F: AACAGAAACTTATTTTCCTGTGT (SEQ ID NO:31) and GJC 106R:GGTCTTCTGCTTGGCTGAGA (SEQ ID NO:32), cloned using TOPO™ (Invitrogen) andsequenced. Similarly, FUT8 intron 9 was PCR amplified from C. griseusgenomic DNA using EasyA™ polymerase (Stratagene) and the oligonucleotideprimers GJC 71F: GCTTGGCTTCAAACATCCAG (SEQ ID. NO:4) and GJC 72R:CACTTTGTCAGTGCGTCTGA (SEQ ID NO:33). The PCR product was cloned andsequenced. The partial sequence of intron 10 was obtained by PCRamplification of C. griseus genomic DNA using EasyA™ polymerase(Stratagene) and the oligonucleotides GJC 75F: AGTCCATGTCAGACGCACTG (SEQID NO:34) and GJC 77R: CAGAACTGTGAGACATAAACTG (SEQ ID NO:35).

The FUT8 cDNA, intron 9 and intron 10 sequences cloned as describedabove were then used for the design of ZFNs binding within FUT8. Inparticular, the FUT8 cDNA (FIG. 1) sequence was scanned for sitesfavorable for recognition by zinc finger nucleases, and one suchlocation that overlapped with the Fut8 enzymatic motif was identified(FIG. 4). In addition, the intronic DNA (FIG. 3) was also scanned forpotential ZFN binding sites.

Several pairs of zinc finger nucleases (ZFNs) were designed to recognizesequences within FUT8. The approximate locations of two ZFN sites in theFUT8 gene are shown in FIG. 4. The sequences of the zinc fingerrecognition helices and the DNA sequences they are designed to recognizeare listed in Table 1. Plasmids comprising sequences encoding FUT8 ZFNswere constructed essentially as described in Umov et al. (2005) Nature435(7042):646-651.

Example 2 Cel-1 Mismatch Assay

To determine whether FUT8-targeted ZFNs modified the endogenous FUT8locus as expected, Cel-1 mismatch assays were performed essentially asper the manufacturer's instructions (Trangenomic SURVEYOR™). Briefly,the appropriate ZFN plasmid pairs were transfected into CHO K-I cells.CHO K-I cells were obtained from the American Type Culture Collectionand grown as recommended in F-12 medium (Invitrogen) supplemented with10% qualified fetal calf serum (FCS, Hyclone). Cells were disassociatedfrom plasticware using TrypLE Select™ protease (Invitrogen). Fortransfection, one million CHO K-I cells were mixed with 1 μg each zincfinger nuclease and 100 μL Amaxa Solution T. Cells were transfected inan Amaxa Nucleofector II™ using program U-23 and recovered into 1.4 mLwarm F-12 medium+10% FCS.

Cells were harvested two days post-transfection and chromosomal DNAprepared using a Masterpure™ DNA Purification Kit (Epicentre). Theappropriate region of the FUT8 locus was PCR amplified using Accuprime™High-fidelity DNA polymerase (Invitrogen). PCR reactions were heated to94° C., and gradually cooled to room temperature. Approximately 200 ngof the annealed DNA was mixed with 0.334 CEL-I™ enzyme (Transgenomic)and incubated for 20 minutes at 42° C. Reaction products were analyzedby polyacrylamide gel electrophoresis in 1× Tris-borate-EDTA buffer.

As shown in FIGS. 5A and 5B, FUT8 ZFNs modified the endogenous FUT8locus. In particular, ZFN pair of ZFN 12029 and ZFN 12030 resulted inmodification of 3.3% of chromosomes (FIG. 5A). ZFN pairs 12170/12176 and12172/12176 modified 3.0% and 4.4% of chromosomes, respectively (FIG.5B).

Example 3 Genotypic Analysis

FUT8 deletion clones were also analyzed at the genetic level. In orderto rapidly identify double-mutant clones, a phenotypic screen based onthe resistance of fucosylation-deficient CHO cells to the lectin Lensculinaris agglutinin (LCA, Vector Laboratories) was used. The CHO cellline Lec13 contains a mutation in the fucose biosynthetic gene GMD thatallows it to grow in concentrations of LCA 50-fold higher than wild-typeCHO cells. See, e.g., Ripka et al. (1986) Somat Cell Mol Genet 12:51-62; Ohyama et al. (1998) J Biol Chem 273:14582-7. FUT8−/− cells failto bind fluorescently-labeled LCA. See, Yamane-Ohnuki et al. (2004)Biotechnol Bioeng 87:614-22. Accordingly, we reasoned that FUT8−/− cellswould also be resistant to growth in LCA.

Cells were transfected as described in Example 2 using the ZFN pair12170/12176, except that between 6 and 30 days post-transfection, theZFN-treated cells were plated into 96-well format at limiting dilution,at approximately 0.4 cells/well. After two weeks of growth the number ofclones per well was scored, the cells washed in lx PBS, and 20 μL TrypLESelect™ (InVitrogen) added. Ten microliters of the disassociated cellswere transferred to parallel 96-well plates containing F-12 medium+10%FCS+50 μg/mL LCA. One-hundred microliters of F-12 medium+10% FCS wasadded to the remaining 10 μL of cells in the original 96-well plate. Themorphology of cells in the LCA-containing plates was scored 18 hourslater. Clones retaining a wild-type non-rounded CHO-KI morphology in thepresence of LCA were noted and the corresponding colony from thenon-LCA-treated plate was expanded. If the original well was found tocontain more than one clone (and also therefore produce a mixture ofrounded and wild-type-appearing cells when grown in LCA), the contentsof the well were redilution cloned as above.

Genomic DNA was harvested from non-LCA-treated LCA-resistant cells and aportion of the FUT8 locus PCR amplified using the oligos GJC 75F:

(SEQ ID NO: 34) AGTCCATGTCAGACGCACTG and GJC 91R: (SEQ ID NO: 7)TGTTACTTAAGCCCCAGGC.

Half of the PCR product (˜200 ng) was analyzed using the CEL-I assay asdescribed above while the other half was gel purified. Purified bandsthat were CEL-I-negative (homozygotes) were sequenced directly.CEL-I-positive bands were Topo®-Cloned (Invitrogen) and clones sequenceduntil two alleles were recovered.

Of the 600 clones analyzed in this manner, 28 were resistant to LCA(4.7%). Fifteen of the 28 LCA-resistant cell lines were single-cellclones. Cell lines were expanded from the half of the culture notexposed to LCA. The FUT8 genotypes of these clones are shown in Table 2.The region of sequence shown here is identical to that shown in FIG. 4.A five base pair gap has been inserted into this depiction of thewild-type sequence to accommodate allelic sequences that contain thesmall insertions found in some clones. Alleles are designated as A andB; clones without allele designations are homozygous. Clone 12-Bcontains a 148 bp insertion of C. griseus ribosomal DNA (rDNA) sequenceas well as the indicated deletion.

TABLE 2 LCA-resistant clones treated with the ZFN pair 12170/12176 ClonePartial Sequence WildAGAGTGTATCTGGCCACTGATGACCCTTCTTT-----GTTAAAGGAGGCAAAGACAAAGTAAGT type(SEQ ID NO: 40) 1-AAGAGTGTATCTGGCCACTGATGACCCTT------------TAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 41) 1-BAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTTA---TAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 42) 2-AAGAGTGTATCTGGCCACTG------------------------------AAAGACAAAGTAAGT(SEQ ID NO: 43) 2-BAGAGTGTATCTGGCCACTGATGACC------------GTTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 44) 3-AAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTTATGTTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 45) 3-BAGAGTGTATCTGGCCACTGATGACCCTTCTTTT----GTTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 46) 4AGAGTGTATCTGGCCACTGATGACCCTTCTTT--------AAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 47) 5AGAGTGNATCTGGCCACTGATGACCCTTCTTT--------AAAGGAGGCAAAGACAANNNANGN(SEQ ID NO: 48) 6AGAGTGTATCTGGCCACTGATGA-----------------------------GACAAAGTAAGT(SEQ ID NO: 49) 7-AAGAGTGTATCTGGCCACTGATGACCCTTCT-------GTTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 50) 7-BAGAGTGTATCTGGCCACTGATGACCCTTCTTT---------AAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 51) 8AGAGTGTATCNGGCCACTGATGACCCTTCTTT--------AAAGGAGGCAAAGACNNAGNAAGT(SEQ ID NO: 52) 9AGAGTGTATCTGGCCACTGATGACCCTTCTTT--------AAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 53) 10-AAGAGTGTATCTG----------------------------------AGCAAAGACAAAGTAAGT(SEQ ID NO: 54) 10-BAGAGTGTATCTGGCCACTG---------------------AAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 55) 11-AAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTT---ATAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 56) 11-BAGAGTGTATCTGGCCACTGATGACCCTT-----------TAA-GGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 57) 12-AAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTTATGGTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 58) 12-BAGAGTGTATCTGGCCACTGAT------(148bp)-----TAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 59) 13-AAGAGTGTATCTGGCCACTGATGACCCTT-----------------------------AGTAAGT(SEQ ID NO: 60) 13-BAGAGTGTATCTGGCCACTGA-------------------------------------------T(SEQ ID NO: 61) 14-AAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTTATGTTAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 62) 14-BAGAGTGTATCTGGCCACTGATGACCCTTCTTTGTT---ATAAAGGAGGCAAAGACAAAGTAAGT(SEQ ID NO: 63) 15AGAGTGTATCTGGCCACTGATGACCCTTCTTT--------AAAGGAGGCNAAGACAGAGTANGT(SEQ ID NO: 64)

For all clones sequenced, both alleles of FUT8 were modified. Five ofthe clones were homozygous. Genotyping also revealed clones withdeletions of between 2 and 38 base pairs and small insertions of 1 to 5base pairs. Allele B of clone 12 contained a 12 base pair deletion aswell as a 148 base pair insertion of C. griseus rDNA sequence.

Example 4 Disruption of FUT8 Via Dual-ZFN-Modification

A larger deletion in FUT8 (1300 bp of FUT8, including the majority ofexon 10) was also created by simultaneous transfection of the intronicZFN pair ZFN 12029/12030 and the exonic pair ZFN 12172/12176. Inparticular, one microgram each of ZFNs 12029, 12030, 12172, and 12176were transfected into CHO K-I cells as described above. Cells wereharvested 2 days post-transfection and the genomic DNA purified. The DNAwas digested with EcoR I and Xmn Ito destroy the wild-type chromosomesand PCR amplified with the oligos GJC 71F:

(SEQ ID NO: 4) GCTTGGCTTCAAACATCCAG  and GJC 91R: (SEQ ID NO: 7)TGTTACTTAAGCCCCAGGC.

Results are shown in FIG. 6A and demonstrate that a fragment of theexpected size was generated.

CHO cell lines were also treated subsequently with different pairs ofZFNs that target the FUT8 gene with or without LCA enrichment. The CEL-Iassay was performed 2 and 30 days post-transfection with ZFN plasmids asdescribed above in Example 2.

As shown in FIG. 6B, LCA treatment resulted in significant increase inthe percentage of FUT8−/− cells.

Example 5 Inactivation of Additional Genes

Cell lines were also created in which FUT8 and additional genes wereinactivated. In particular, zinc finger nucleases directed to DHFR andGS were designed and constructed as described in U.S. Patent PublicationNos. 2006/0063231 and U.S. 2008/015164. Plasmids encoding DHFR- andGS-targeted ZFNs were introduced into CHO cells as described in Example2 to create GS^(−/−)/DHFR^(−/−) CHO cell line.

The GS^(−/−)/DHFR^(−/−) CHO cell lines were subsequently treated witheither of four different pairs of ZFNs that target the FUT8 gene (pools1, 3, 5, and 7). Each pool was subjected to LCA to select for thepopulation in which FUT8 expression had been destroyed (FIG. 7, With LCAenrichment). The CEL-I assay was performed on both the LCA-selected andunselected (FIG. 7, no LCA enrichment) pools as described above.

As shown in FIG. 7, the frequency of disrupted copies of the FUT8 genein LCA-selected pools was as high as 34% (pool 1 with LCA enrichment).

Genotyping analysis of the various triple knockout clones isolated frompool #1 or pool #5 was also performed, essentially as described inExample 3 above. As shown in FIG. 8, of the 75 clones screened from pool#1, 33 (or ˜44%) were modified at both copies of the FUT8 gene.

Finally, CHO cells in which DHFR, GS and FUT8 were disrupted bytreatment with ZFNs were also tested for their ability to bindfluorescent LCA. Approximately 100,000 cells were trypsinized, washed in1×PBS, and mixed with 2 μg/mL fluorescein-LCA (F-LCA). F-LCA binding wasassayed by flow cytometery (Yamane-Ohnuki et al. (2004) Biotech. Bioeng.87:614). As shown in FIG. 9, fluorescent LCA does not bind to cells inwhich GS, DHFR and FUT8 genes are disrupted. Thus, cells in which any ofFUT8 and one, two (or more) genes are inactivated are used forexpression (over-expression) of recombinant proteins of interest.

These results show the rapid generation of Fut8-deficient cell linesusing ZFNs targeted to cleave the FUT8 gene. DNA repair through theerror-prone process of NHEJ at the site of cleavage resulted infunctionally deleterious mutations. Although NHEJ-derived mutations aresometimes small relative to those made by conventional gene disruption,the ability of ZFNs described herein to target these mutations to theDNA coding for the critical catalytic region of FUT8 ensured that evensmall, in-frame alleles would result in severe defects in enzymeactivity. For example, homozygous deletion of only leucine 413 (clones5, 8, 9, and 15) resulted in cells resistant to LCA.

Furthermore, although many different subtypes of CHO cell lines exist,often with custom-made genetic or phenotypic changes, the ZFNs describedherein can be used to rapidly disrupt FUT8 in any cell line or subtype.In addition, because zinc finger protein binding sites can be selectedthat are conserved between mammalian species, ZFNs can be designed toinactive FUT8 in cells lines derived from any species.

Example 6 Fut8 Hypomorphs

Some CHO cells with ZFN modification of FUT8 may retain a fraction oftheir wild-type Fut8 activity. Such cells might be resistant to therelatively low concentration of LCA used to perform the initial screen(50 μg/mL) but remain sensitive to higher concentrations of LCA.

Cells were transfected as described in Example 2 and screened forresistance to 50 μg/mL LCA and genotyped as in Example 3. A CEL-I assayof individual clones and the genotypes of some of these clones are shownin FIG. 10. After this primary screen with 50 μg/mL LCA, a secondaryscreen of these initial ZFN-modified LCA-resistant cell lines withhigher concentrations of LCA was performed to identify hypomorphs. Celllines resistant to 50 μg/mL LCA were assayed for growth in 100, 200,400, and 800 μg/mL LCA. Eleven of the 16 cell lines tested in thismanner exhibited wild-type growth and cell morphology at 800 μg/mL LCA.Five of the 16 cell lines tested exhibited wild-type growth and cellmorphology only at LCA concentrations below 800 μg/mL These five cloneswith intermediate LCA resistance are therefore hypomorphic for Fut8activity. Fut8 hypomorphism perfectly correlated with the presence of athree nucleotide (ATT) insertion between the ZFN binding sites. Thisinsertion adds one leucine residue to the C. griseus Fut8 protein atposition 415. The other allele in each of these clones is predicted toeliminate enzyme activity. Two of these five clones are clone 14 andclone 19 and are shown in FIG. 10.

The hypomorphs discovered in the LCA-resistance titration were confirmedby assay of fluorescent-LCA (F-LCA) binding to cell surface-exposedα1-6-linked fucose. For each cell line analyzed approximately 100,000cells were trypsinized, washed in 1×PBS, and mixed with 2 μg/mLfluorescein-LCA (F-LCA). F-LCA binding was assayed by flow cytometery(Guava Technologies). All of the hypomorphic clones examined by F-LCAbinding had ˜5-fold less F-LCA binding than wild-type; all clonesresistant to 800 ng/mL LCA showed no ability to bind F-LCA. Staining ofwild-type CHO-KI cells and one such hypomorphic clone is shown in FIG.11.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method of inactivating an endogenous cellular FUT8 gene in a cell, the method comprising: introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises: (a) a first zinc finger DNA-binding domain, wherein the DNA-binding domain comprises four or five zinc finger recognition regions ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus, and wherein F1 to F4, F1 to F5 or F1 to F6 comprise amino acid sequences selected from the group consisting of: (i) F1: QSSDLSR (SEQ ID NO: 9); F2: TSGNLTR (SEQ ID NO: 10); F3: RSDDLSK (SEQ ID NO: 11); and F4: DRSALAR (SEQ ID NO: 12); (ii)  F1: RSDVLSA (SEQ ID NO: 14); F2: QNATRIN (SEQ ID NO: 15); F3: DRSNLSR (SEQ ID NO: 16); and F4: RLDNRTA (SEQ ID NO: 17); (iii) F1: RSDNLRE (SEQ ID NO: 26); F2: NNTQLIE (SEQ ID NO: 27); F3: TSSILSR (SEQ ID NO: 28); F4 RSDNLSA (SEQ ID NO: 29); and F5: RKDTRIT (SEQ ID NO: 30); and (iv)  F1: RSDNLSV (SEQ ID NO: 19); F2: QNATRIN (SEQ ID NO: 15); F3: RSDNLST (SEQ ID NO: 20)  or RSDHLSQ (SEQ ID NO: 24); F4: QSATRTK (SEQ ID NO: 21); F5 RSDNLSR (SEQ ID NO: 22); and F6: RNDNRKT (SEQ ID NO: 23); and

(b) a first cleavage domain; such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the FUT8 gene; and introducing a nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises: (i) a second zinc finger DNA-binding domain that is engineered to bind to a second target site in the FUT8 gene; and (ii) a second cleavage domain; such that the second polypeptide is expressed in the cell, whereby the first and second polypeptides bind to their respective target sites, dimerize and cleave the FUT8 gene, thereby inactivating the FUT8 gene.
 2. The method of 1, wherein the first and second polypeptides are encoded by the same nucleic acid.
 3. The method of claim 1, wherein the first and second polypeptides are encoded by different nucleic acids.
 4. The method of claim 1, wherein the first or second cleavage domain is a wild-type Fold cleavage domain.
 5. The method of claim 1, wherein the first or second cleavage domain is an engineered FokI cleavage domain.
 6. The method of claim 1, further comprising introducing donor sequence into the cell such that the donor sequence is integrated into the inactivated FUT8 gene.
 7. A method of producing a recombinant protein of interest in a host cell, the method comprising the steps of: (a) providing a host cell comprising an endogenous FUT8 gene; (b) inactivating the endogenous FUT8 gene of the host cell by the method of claims 1; and (c) introducing an expression vector comprising a transgene, the transgene comprising a sequence encoding a protein of interest into the host cell, thereby producing the recombinant protein.
 8. The method of claim 7, wherein the protein of interest comprises an antibody.
 9. A cell or cell line in which Fut8 is partially or fully inactivated, wherein the cell or cell line is produced by (a) inactivating Fut8 in a cell according to the method of claim 1; and (b) culturing the cell under conditions suitable for generating a cell or cell line in which Fut8 is partially or fully inactivated.
 10. The cell or cell line of claim 9, wherein the cell is a mammalian cell or cell line selected from the group consisting of a COS cell, a CHO cell, a VERO cell, a MDCK cell, a WI38 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NS0 cell, a SP2/0-Ag14 cell, a HeLa cell, an HEK293 cell, and a perC6 cell.
 11. The cell or cell line of claim 9, wherein one or more additional genes are partially or fully inactivated.
 12. The cell or cell line of claim 11, wherein dihydrofolate reducatase (DHFR) and glutamine synthetase (GS) have been inactivated. 